This invention pertains generally to the field of plasma processing of materials and to the functionalization of organic and inorganic polymeric substrates.
Molecular recognition and molecular manufacturing systems developed from immobilized biomolecules (enzymes, oligonucleotides, cells) will play an essential role in advanced future technologies and medicine. The anchorage of bioactive molecules on specific substrate surfaces is the key element in the development of molecular assemblers. The inertness of the substrates, the nature of linkages between the substrates and biomolecules, the topographies of the host-surfaces, and the xe2x80x9cchained-freedomxe2x80x9d of the active molecules are characteristics which can be achieved only with difficulty using conventional chemistry approaches.
Molecular recognition between biological macromolecules, small molecules, macromolecules and specific surfaces plays a crucial role in the understanding of various biological systems and in the design of artificial, intelligent surfaces (molecular manufacturing systems) with tremendous practical potentials. As soon as a primitive molecular assembler which is able to self-replicate by atomic-precision-positional-control is constructed, migration pathways will allow the generation of increasingly sophisticated assemblers.
To understand the molecular basis of the interaction, controlled immobilization of biomolecules must be performed by taking into account the chemical and physical (crystallinity, morphology) structure of the substrates, the nature of the bonding between the biologically active molecules and substrates, the surface density of immobilized molecules, and the distance between the anchored biomolecules and the substrate. By understanding and controlling surface functionalization and the consequent anchoring reaction mechanisms, tailored and very specific reaction pathways can be developed (molecular recognition).
Polymer-bound oligonucleotides will find their applications in hybridization-based diagnostics and in the discovery of new therapeutics based on molecular recognition. Prenatal diagnostics of genetic aberrations, identification of virus born diseases, detection of mutations of regulatory proteins controlling carcinogenesis, and novel hybridization-based identification techniques oriented to forensic or archaeology fields are some of the potential applications.
Molecular recognition will also play an essential role in areas other than medicine, pharmaceutics and biotechnology. Development of ultra-selective chemical sensors and absorbent surfaces are crucial for creating environmentally safe processes. Monitoring the quality of water is one of the major demands in this area. Biomolecular-based chemical sensors and filters for toxic chemicals and microorganisms (e.g. E. coli) will play a significant role in future technologies.
Most of the natural and synthetic polymeric substrates can easily be functionalized through polymer-analog reactions. Main chain and side-group homogeneous reactions are the most common approaches. The use of polymers as xe2x80x9ccarriersxe2x80x9d or xe2x80x9csupportsxe2x80x9d for chemical reagents, catalysts or substrates represents a relatively new, significant, and rapidly developing area. The polymer is in the form of an insoluble, inert substrate that may be a solvent-swollen, crosslinked gel, or a surface active solid. This approach eases the separation of reagents or catalysts (e.g. enzymes) from the reaction products, permitting consequently the automation of the complex chemistry. However, the specific structure of the repeating units of the macromolecules often limit considerably the variety of polymer-analog reactions. These reactions are even more difficult to develop under heterogeneous environments. Natural and even some synthetic polymeric substrates can also undergo undesired chemical modifications, and sometimes biodegradation, during the polymer-supported organic reactions. Inert polymeric substrates (e.g. polyethylene, polypropylene, PET, PTFE) and inorganic supports (e.g. glass, silica) however, cannot be functionalized efficiently by using conventional wet chemistry approaches.
Cold plasma processing has shown promise for the functionalization of organic and inorganic substrates. See, e.g., D. T. Clark, et al., Polymer Surfaces (book), John Wiley and Sons, New York, 1978, pp. 185-210; F. Denes, et al.,xe2x80x9cSurface Modification of Polysaccharides Under Cold Plasma Conditions,xe2x80x9d in Polysaccharides. Structural Diversity and Functional Versatility (book), Ed. S. Dumitriu, Marcel Dekker, Inc., New York, 1998; Plasma Surface Modification of Polymers: Relevance to Adhesion (book), Eds. M. Strobel, et al., VSP, Utrecht, The Netherlands, 1994; F. Denes, TRIP, Vol., No. 1, 1997, pp. 23, et seq. Numerous experiments performed in recent years in plasma laboratories under various internal and external plasma conditions and reactor geometries clearly indicate that inert and reactive-gas discharges are effective for the surface modification (functionalization) of even the most inert materials, such as polypropylene, Teflon(copyright), silica, etc. The industrial applications of macromolecular plasma chemistry are rapidly developing. Large capacity reactors and continuous flow system plasma installations have been designed, developed and tested.
The most widely used synthetic polymer surfaces are usually characterized by low surface energy values, and some of the thermoplastics, including polyethylene and polypropylene, for example, are essentially chemically inert. Modification of characteristics like adhesion, wettability, dyeability, and reactivity for such materials necessitates the creation of particular functionalities on the surfaces of such polymer substrates.
The advantages of using enzymes in chemical synthesis are related to their very high specificity (regio- and stereo-specificity) and versatility, mild reaction conditions (close to room temperatures and to pH neutral media), and to their high reaction rates. However, due to the poor recovery yields and reusability of free enzymes, much attention has been paid in the last few years to the development of efficient enzyme immobilization processes. Most biologically-active in vivo species, such as enzymes and antibodies, function in heterogeneous media. These environments are difficult to reproduce in vitro for industrial utilization. Immobilized enzyme systems are useful for experimental and theoretical research purposes for understanding the mechanisms of in vivo, bio-catalysed reactions, and offer solutions for use in batch-type reactions, where there is poor adaptability to various technological designs and recovery of the enzymes is difficult.
The molecular recognition ability and activity of enzymes (polypeptide molecules) are based on their complex three-dimensional structures containing sterically exposed, specific functionalities. The polypeptide chains are folded into one or several discrete units (domains), which represent the basic functional and three-dimensional structural entities. The cores of domains are composed of a combination of motifs which are combinations of secondary structure elements with a specific geometric arrangement. The molecular-structure-driven chain-folding mechanisms generate three-dimensional enzyme structures with protein molecules orienting their hydrophobic side chains toward the interior and exposing a hydrophilic surface. The xe2x80x94C(R)xe2x80x94COxe2x80x94NHxe2x80x94 based main chain is also organized into a secondary structure to neutralize its polar components through hydrogen bonds. These structural characteristics are extremely important and they make the enzyme molecules very sensitive to the morphological and functional characteristics of the potential immobilizing substrates. High surface-concentrations of enzyme-anchoring functionalities can result, for instance, in excessive enzyme-desities or multi-point connections which can xe2x80x9cneutralizexe2x80x9d the active sites or can alter the three-dimensional morphologies of the enzyme molecules through their mutual interaction and their interaction with the substrate surfaces. These are just a few of the factors which may be responsible for the significantly lower activities of immobilized-enzymes in comparison to the activities of free enzyme molecules. Rough substrate surface topographies or stereoregular surfaces (e.g., isotactic or syndiotactic polymers) might also influence, in a positive or negative way, the specific activities. Morphologically ordered surfaces might induce changes of the stereoregular shapes of protein molecules. It has also been found that enzymes can adopt more than one functional conformation other than its lowest potential energy state. E. S. Young, et al., Anal. Chem. Vol. 69, 1977, pp. 4242, et seq.
In accordance with the invention, silicon-chlorine atoms-based functionalities are implanted in substrates by exposing the substrates to a cold plasma of a gas selected from dichlorosilane, silicon tetrachloride, hexachlorodisilane and mixtures thereof. Various inorganic substrates such as glass and silica and organic substrates (e.g., polymers such as polyethylene, polycarbonate, polystyrene, etc.) may be efficiently functionalized in this manner. The substrates having plasma implanted surface functionalities can then be used to initiate gas phase in situ derivatization reactions to form linker molecules attached to the substrate. The reaction is carried out within the same reactor to avoid exposure of the functionalized surface to unwanted materials. After the plasma is terminated, the reactor reaction chamber is evacuated to remove the plasma forming gas and a selected reactant gas is introduced into the chamber and exposed to the substrate. Various suitable reactant gases may be utilized, examples of which include di-acylchloride derivatives, diamines and anhydrides, such as: ethlylenediamine, propylenediamine, hexafluoro 1,3 propylenediamine, pentafluoropropionic anhydride, hexafluoroglutaric anhydride, etc. The gas phase reaction may be carried out in multiple steps to provide a linked chain of spacer molecules of a desired length and structure. These spacer molecules are available for binding to various other reactants, particularly biomolecules including enzymes and nucleotides. For example, an enzyme that becomes bound to the substrate by the strands of spacer molecules can retain freedom of movement and conformation that is comparable to that of the free enzyme. In this manner, the activity of bioactive molecules such as enzymes and nucleotides can be significantly enhanced over that of such molecules bound directly to the substrate.
Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings.